Proton‐dependent zinc release from intracellular ligands

Abstract

In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn2+ into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn2+. Although exposure to the protonophore FCCP precludes re-loading of the mitochondria and acidic organelles with Zn2+, FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn2+. Therefore, Zn2+-releasing stores were not mitochondria or acidic organelles but rather intracellular Zn2+ ligands. To test which ligands might be involved, the rate of acid-induced Zn2+ release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn2+] was monitored in vitro using the ratiometric Zn2+-sensitive fluorescent probe FuraZin-1. Carnosine failed to chelate Zn2+ but did chelate Cu2+; the remaining ligands chelated Zn2+ and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes rapidly accelerated the rate of Zn2+ release. The zinc-cysteine complexes also released Zn2+ when a histidine-modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc-cysteine complexes can contain micromolar amounts of Zn2+, these complexes may represent the stores responsible for an acid-induced intracellular Zn2+ release.

Intracellular zinc activates KCNQ channels by reducing their dependence on phosphatidylinositol 4,5-bisphosphate

“…Cytosolic free zinc levels at rest are too low to affect KCNQ channel activity (<1 nM) (12); these low levels likely explain the observation that, although TPEN reversed the augmentation of the KCNQ current by exogenous zinc, it rarely reduced the amplitude of KCNQ channels overexpressed in nonexcitable CHO cells to levels below the baseline. However, zinc can reach millimolar levels in the synaptic vesicles of neurons (13); another releasable pool of zinc is bound to intracellular proteins such as metallothioneins (15). Vesicular zinc is released during synaptic transmission and loads postsynaptic terminals through zincpermeable channels (e.g., AMPA) (14).…”

“…Vesicular zinc is released during synaptic transmission and loads postsynaptic terminals through zincpermeable channels (e.g., AMPA) (14). Zinc also can be released from cytosolic buffers in response to acidification (15), as observed in hypoxic conditions. In a recent study FluoZin3 zinc imaging revealed large intracellular zinc transients (comparable to the rises recorded in this study in response to zinc ionophores) in hippocampal pyramidal neurons in response to oxidative stress (14).…”

“…During synaptic transmission vesicular zinc is released, and quantities of zinc translocate to the postsynaptic terminals through zinc-permeable channels such as AMPA receptors (14). Additionally, zinc can be released from cytosolic buffering proteins such as metallothioneins, e.g., in response to acidification (15). Thus, there is clear scope for intracellular zinc signaling in neurons, including postsynaptic rises during periods of high activity, hypoxia, neurotrauma, and other conditions.…”

Neuronal acid‐induced [Zn2+]i elevations calibrated using the low‐affinity ratiometric probe FuraZin‐1

“…However, neurotoxic phenomena associated with ischemia/reperfusion, such as oxidative stress and acidification, lead to Zn 2+ release from intracellular stores (Aizenman et al 2000; Sensi et al 2003). For example, a drop in intracellular pH (pH i ) below 6.5 causes Zn 2+ release from intracellular cysteine-containing ligands; yet some intracellular ligands bind Zn 2+ when pH i drops to 6.1 (Kiedrowski 2014). ATP might be one such ligand because it binds Zn 2+ (Churchich et al 1989; Nakano and McCormick 1991) and its intracellular concentration in the brain is in the millimolar range (Erecinska and Silver 1989).…”

“…However, for routine studies aimed to determine the impact of various experimental conditions on [Zn 2+ ] i , this approach is not practical. Therefore in the present work, an attempt was made to calibrate acid-induced [Zn 2+ ] i elevations using a low affinity ratiometric Zn 2+ probe, FuraZin-1, whose Zn 2+ K d at pH 6.1 is 39 μM (Kiedrowski 2014). Contrary to the previous estimates obtained using high affinity Zn 2+ probes, the data obtained using FuraZin-1 show that, upon acid-induced Zn 2+ release from intracellular stores, [Zn 2+ ] i elevates to about 2 μM.…”